This invention relates to the field of receptors belonging to the superfamily of nuclear hormone receptors, in particular to steroid receptors. The invention relates to DNA encoding a novel steroid receptor, the preparation of said receptor, the receptor protein, and the uses thereof.
Steroid hormone receptors belong to a superfamily of nuclear hormone receptors involved in ligand-dependent transcriptional control of gene expression. In addition, this superfamily consists of receptors for non-steroid hormones such as vitamine D, thyroid hormones and retinoids (Giguxc3xa8re et al, Nature 330, 624-629, 1987; Evans, R. M., Science 240, 889-895,1988). Moreover, a range of nuclear receptor-like sequences have been identified which encode socalled xe2x80x98orphanxe2x80x99 receptors: these receptors are structurally related to and therefore classified as nuclear receptors, although no putative ligands have been identified yet (B. W. O""Malley, Endocrinology 125, 1119-1170, 1989; D. J. Mangelsdorf and R. M. Evans, Cell, 83, 841-850, 1995).
The superfamily of nuclear hormone receptors share a modular structure in which six distinct structural and functional domains, A to F, are displayed (Evans, Science 240, 889-895, 1988). A nuclear hormone receptor is characterized by a variabel N-terminal region (domain A/B), followed by a centrally located, highly conserved DNA-binding domain (hereinafter referred to as DBD; domain C), a variable hinge region (domain D), a conserved ligand-binding domain (herein after referred to as LBD; domain E) and a variable C-terminal region (domain F).
The N-terminal region, which is highly variable in size and sequence, is poorly conserved among the different members of the superfamily. This part of the receptor is involved in the modulation of transcription activation (Bocquel et al, Nucl. Acid Res., 17, 2581-2595, 1989; Tora et al, Cell 59, 477-487, 1989).
The DBD consists of approximately 66 to 70 amino acids and is responsible for DNA-binding activity: it targets the receptor to specific DNA sequences called hormone responsive elements (hereinafter referred to as HRE) within the transcription control unit of specific target genes on the chromatin (Martinez and Wahli, In xe2x80x98Nuclear Hormone Receptorsxe2x80x99, Acad. Press, 125-153, 1991).
The LBD is located in the C-terminal part of the receptor and is primarily responsible for ligand binding activity. In this way, the LBD is essential for recognition and binding of the hormone ligand and, in addition possesses a transcription activation function, thereby determining the specificity and selectivity of the hormone response of the receptor. Although moderately conserved in structure, the LBD""s are known to vary considerably in homology between the individual members of the nuclear hormone receptor superfamily (Evans, Science 240, 889-895, 1988; P. J. Fuller, FASEB J., 5, 3092-3099, 1991; Mangelsdorf et al, Cell, Vol. 83, 835-839, 1995).
Functions present in the N-terminal region, LBD and DBD operate independently from each other and it has been shown that these domains can be exchanged between nuclear receptors (Green et al, Nature, Vol. 325, 75-78, 1987). This results in chimeric nuclear receptors, such as described for instance in WO-A-8905355.
When a hormone ligand for a nuclear receptor enters the cell by diffusion and is recognized by the LBD, it will bind to the specific receptor protein, thereby initiating an allosteric alteration of the receptor protein. As a result of this alteration the ligand/receptor complex switches to a transcriptionally active state and as such is able to bind through the presence of the DBD with high affinity to the corresponding HRE on the chromatin DNA (Martinez and Wahli, xe2x80x98Nuclear Hormone Receptorsxe2x80x99, 125-153, Acad. Press, 1991). In this way the ligand/receptor complex modulates expression of the specific target genes. The diversity achieved by this family of receptors results from their ability to respond to different ligands.
The steroid hormone receptors are a distinct class of the nuclear receptor superfamily, characterized in that the ligands are steroid hormones. The receptors for glucocorticoids (GR), mineralcorticoids (MR), progestins (PR), androgens (AR) and estrogens (ER) are classical steroid receptors. Furthermore, the steroid receptors have the unique ability upon activation to bind to palindromic DNA sequences, the so-called HRE""s, as homodimers. The GR, MR, PR and AR recognize the same DNA sequence, while the ER recognizes a different DNA sequence. (Beato et al, Cell, Vol. 83, 851-857, 1995). After binding to DNA, the steroid receptor is thought to interact with components of the basal transcriptional machinery and with sequence-specific transcription factors, thus modulating the expression of specific target genes.
Several HRE""s have been identified, which are responsive to the hormone/receptor complex. These HRE""s are situated in the transcriptional control units of the various target genes such as mammalian growth hormone genes (responsive to glucocorticoid, estrogen, testosterone), mammalian prolactin genes and progesterone receptor genes (responsive to Estrogen), avian ovalbumin genes (responsive to progesterone), mammalian metallothionein gene (responsive to glucocorticoid) and mammalian hepatic xcex12xcexc-globulin gene (responsive to estrogen, testosterone, glucocorticoid).
The steroid hormone receptors have been known to be involved in embryonic development, adult homeostasis as well as organ physiology. Various diseases and abnormalities have been ascribed to a disturbance in the steroid hormone pathway. Since the steroid receptors exercise their influence as hormone-activated transcriptional modulators, it can be anticipated that mutations and defects in these receptors, as well as overstimulation or blocking of these receptors might be the underlying reason for the altered pattern. A better knowledge of these receptors, their mechanism of action and of the ligands which bind to said receptor might help to create a better insight in the underlying mechanism of the hormone signal transduction pathway, which eventually will lead to better treatment of the diseases and abnormalities linked to altered hormone/receptor functioning.
For this reason cDNA""s of the steroid and several other nuclear receptors of several mammalians, including humans, have been isolated and the corresponding amino acid sequences have been deduced, such as for example the human steroid receptors PR, ER, GR, MR, and AR, the human non-steroid receptors for vitamine D, thyroid hormones, and retinoids such as retinol A and retinoic acid. In addition, cDNA""s encoding well over 100 mammalian orphan receptors have been isolated, for which no putative ligands are known yet (Mangelsdorf et al, Cell, Vol.83, 835-839, 1995). However, there is still a great need for the elucidation of other nuclear receptors, in order to unravel the various roles these receptors play in normal physiology and pathology.
The present invention provides for such a novel nuclear receptor. More specific, the present invention provides for novel steroid receptors, having estrogen mediated activity. Said novel steroid receptors are novel estrogen receptors, which are able to bind and be activated by, for example, estradiol, estrone and estriol.
According to the present invention it has been found that a novel estrogen receptor is expressed as an 8 kb transcript in human thymus, spleen, peripheral blood lymphocytes (PBLs), ovary and testis. Furthermore, additional transcripts have been identified. Another transcript of approximately 10 kb was identified in ovary, thymus and spleen. In testis, an additional transcript of 1.3 kb was detected. These transcripts are probably generated by alternative splicing of the gene encoding the novel estrogen receptor according to the invention.
Cloning of the cDNA""s encoding the novel estrogen receptors according to the invention revealed that several splicing variants of said receptor can be distinguished. At the protein level, these variants differ only at the C-terminal part.
cDNA encoding an ER has been isolated (Green, et al, Nature 320, 134-139, 1986; Greene et al, Science 231, 1150-1154, 1986), and the corresponding amino acid sequence has been deduced. This receptor and the receptor according to the present invention, however, are distinct, and encoded for by different genes with different nucleic acid sequences. Not only do the ER of the prior art (hereinafter referred to as classical ER) and the ER according to the present invention differ in amino acid sequence, they also are located on different chromosomes. The gene encoding the classical ER is located on chromosome 6, whereas the gene encoding the ER according to the invention was found to be located on chromosome 14. The ER according to the invention furthermore distinguishes itself from the classical receptor in differences in tissue distribution, indicating that there may be important differences between these receptors at the level of estrogenic signalling.
In addition, two orphan receptors, ERRxcex1 and ERRxcex2, having an estrogen receptor related structure have been described (Giguxc3xa8re et al, Nature 331, 91-94, 1988). These orphan receptors, however, have not been reported to be able to bind estrodial or any other hormone that binds to the classical ER, and other ligands which bind to these receptors have not been found yet. The novel estrogen receptor according to the invention distinguishes itself clearly from these receptors since it was found to bind estrogens.
The fact that a novel ER according to the invention has been found is all the more surprising, since any suggestion towards the existence of additional estrogen receptors was absent in the scientific literature: neither the isolation of the classical ER nor the orphan receptors ERRxcex1 and ERRxcex2 suggested or hinted towards the presence of additional estrogen receptors such as the receptors according to the invention. The identification of additional ER""s could be a major step forward for the existing clinical therapies, which are based on the existence of one ER and as such ascribe all estrogen mediated abnormalities and/or diseases to this one receptor. The receptors according to the invention will be useful in the development of hormone analogs that selectively activate either the classical ER or the novel estrogen receptor according to the invention. This should be considered as one of the major advantages of the present invention.
Thus, in one aspect, the present invention provides for isolated cDNA encoding a novel steroid receptor. In particular, the present invention provides for isolated cDNA encoding a novel estrogen receptor.
According to this aspect of the present invention, there is provided an isolated DNA encoding a steroid receptor protein having an N-terminal domain, a DNA-binding domain and a ligand-binding domain, wherein the amino acid sequence of said DNA-binding domain of said receptor protein exhibits at least 80% homology with the amino acid sequence shown in SEQ ID NO:3, and the amino acid sequence of said ligand-binding domain of said receptor protein exhibits at least 70% homology with the amino acid sequence shown in SEQ ID NO:4.
In particular, the isolated DNA encodes a steroid receptor protein having an N-terminal domain, a DNA-binding domain and a ligand-binding domain, wherein the amino acid sequence of said DNA-binding domain of said receptor protein exhibits at least 90%, preferably 95%, more preferably 98%, most preferably 100% homology with the amino acid sequence shown in SEQ ID NO:3.
More particularly, the isolated DNA encodes a steroid receptor protein having an N-terminal domain, a DNA-binding domain and a ligand-binding domain , wherein the amino acid sequence of said ligand-binding domain of said receptor protein exhibits at least 75%, preferably 80%, more preferably 90%, most preferably 100% homology with the amino acid sequence shown in SEQ ID NO:4.
A preferred isolated DNA according to the invention encodes a steroid receptor protein having the amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:21 or SEQ ID NO:25.
A more preferred isolated DNA according to the invention is an isolated DNA comprising a nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:20 or SEQ ID NO:24.
The DNA according to the invention may be obtained from cDNA. Alternatively, the coding sequence might be genomic DNA, or prepared using DNA synthesis techniques.
The DNA according to the invention will be very useful for in vivo expression of the novel receptor proteins according to the invention in sufficient quantities and in substantially pure form.
In another aspect of the invention, there is provided for a steroid receptor comprising the amino acid sequence encoded by the above described DNA molecules.
The steroid receptor according to the invention has an N-terminal domain, a DNA-binding domain and a ligand-binding domain, wherein the amino acid sequence of said DNA-binding domain of said receptor exhibits at least 80% homology with the amino acid sequence shown in SEQ ID NO:3, and the amino acid sequence of said ligand-binding domain of said receptor exhibits at least 70% homology with the amino acid sequence shown in SEQ ID NO:4.
In particular, the steroid receptor according to the invention has an N-terminal domain, a DNA-binding domain and a ligand-binding domain, wherein the amino acid sequence of said DNA-binding domain of said receptor exhibits at least 90%, preferably 95%, more preferably 98%, most preferably 100% homology with the amino acid sequence shown in SEQ ID NO:3.
More particular, the steroid receptor according to the invention has an N-terminal domain, a DNA-binding domain and a ligand-binding domain, wherein the amino acid sequence of said ligand-binding domain of said receptor exhibits at least 75%, prefearbly 80%, more preferably 90%, most preferably 100% homology with the amino acid sequence shown in SEQ ID NO:4.
It will be clear for those skilled in the art that also steroid receptor proteins comprising combined DBD and LBD preferences and DNA encoding such receptors are subject of the invention.
Preferably, the steroid receptor according to the invention comprises an amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:21 or SEQ ID NO:25.
Also within the scope of the present invention are steroid receptor proteins which comprise variations in the amino acid sequence of the DBD and LBD without loosing their respective DNA-binding or ligand-binding activities. The variations that can occur in those amino acid sequences comprise deletions, substitutions, insertions, inversions or additions of (an) amino acid(s) in said sequence, said variations resulting in amino acid difference(s) in the overall sequence. It is well known in the art of proteins and peptides that these amino acid differences lead to amino acid sequences that are different from, but still homologous with the native amino acid sequence they have been derived from.
Amino acid substitutions that are expected not to essentially alter biological and immunological activities, have been described in for example Dayhof, M. D., Atlas of protein sequence and structure, Nat. Biomed. Res. Found., Washington D.C., 1978, vol. 5, suppl. 3. Amino acid replacements between related amino acids or replacements which have occurred frequently in evolution are, inter alia Ser/Ala, Ser/Gly, Asp/Gly, Arg/Lys, Asp/Asn, Ile/Val. Based on this information Lipman and Pearson developed a method for rapid and sensitive protein comparison (Science 227, 1435-1441, 1985) and determining the functional similarity between homologous polypeptides.
Variations in amino acid sequence of the DBD according to the invention resulting in an amino acid sequence that has at least 80% homology with the sequence of SEQ ID NO:3 will lead to receptors still having sufficient DNA binding activity. Variations in amino acid sequence of the LBD according to the invention resulting in an amino acid sequence that has at least 70% homology with the sequence of SEQ ID NO:4 will lead to receptors still having sufficient ligand binding activity.
Homology as defined herein is expressed in percentages, determined via PCGENE. Homology is calculated as the percentage of identical residues in an alignment with the sequence according to the invention. Gaps are allowed to obtain maximum alignment.
Comparing the amino acid sequences of the classical ER and the ER""s according to the invention revealed a high degree of similarity within their respective DBD""s. The conservation of the P-box (amino acids E-G-X-X-A) which is responsible for the actual interactions of the classical ER with the target DNA element (Zilliacus et al., Mol.Endo. 9, 389, 1995; Glass, End.Rev. 15, 391, 1994), is indicative for a recognition of estrogen responsive elements (ERE""s) by the ER""s according to the invention. The receptors according to the invention indeed showed ligand-dependent transactivation on ERE-containing reporter constructs. Therefore, the classical ER and the novel ER""s according to the invention may have overlapping target gene specificities. This could indicate that in tissues which co-express both respective ER""s, these receptors compete for ERE""s. The ER""s according to the invention may regulate transcription of target genes differently from classical ER regulation or could simply block classical ER functioning by occupying estrogen responsive elements. Alternatively, transcription might be influenced by heterodimerization of the different receptors.
Thus, a preferred steroid receptor according to the invention comprises the amino acid sequence E-G-X-X-A within the P box of the DNA binding domain, wherein X stands for any amino acid. Also within the scope of the invention is isolated DNA encoding such a receptor.
Methods to prepare the receptors according to the invention are well known in the art (Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989). The most practical approach is to produce these receptors by expression of the DNA encoding the desired protein.
A wide variety of host cell and cloning vehicle combinations may be usefully employed in cloning the nucleic acid sequence coding for the receptor of the invention. For example, useful cloning vehicles may include chromosomal, non-chromosomal and synthetic DNA sequences such as various known bacterial plasmids and wider host range plasmids and vectors derived from combinations of plasmids and phage or virus DNA. Useful hosts may include bacterial hosts, yeasts and other fungi, plant or animal hosts, such as Chinese Hamster Ovary (CHO) cells or monkey cells and other hosts.
Vehicles for use in expression of the ligand-binding domain of the present invention will further comprise control sequences operably linked to the nucleic acid sequence coding for the ligand-binding domain. Such control sequences generally comprise a promoter sequence and sequences which regulate and/or enhance expression levels. Furthermore an origin of replication and/or a dominant selection marker are often present in such vehicles. Of course control and other sequences can vary depending on the host cell selected.
Techniques for transforming or transfecting host cells are quite known in the art (see, for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1989).
Recombinant expression vectors comprising the DNA of the invention as well as cells transformed with said DNA or said expression vector also form part of the present invention.
In a further aspect of the invention, there is provided for a chimeric receptor protein having an N-terminal domain, a DNA-binding domain, and a ligand-binding domain, characterized in that at least one of the domains originates from a receptor protein according to the invention, and at least one of the other domains of said chimeric protein originates from another receptor protein from the nuclear receptor superfamily, provided that the DNA-binding domain and the ligand-binding domain of said chimeric receptor protein originate from different proteins.
In particular, the chimeric receptor according to the invention comprises the LBD according to the invention, said LBD having an amino acid sequence which exhibits at least 70% homology with the amino acid sequence shown in SEQ ID NO:4. In that case the N-terminal domain and DBD should be derived from another nuclear receptor, such as for example PR. In this way a chimeric receptor is constructed which is activated by a ligand of the ER according to the invention and which targets a gene under control of a progesterone responsive element. The chimeric receptors having a LBD according to the invention are useful for the screening of compounds to identify novel ligands or hormone analogs which are able to activate an ER according to the invention.
In addition, chimeric receptors comprising a DBD according to the invention, said DBD having an amino acid sequence exhibiting at least 80% homology with the amino acid sequence shown in SEQ ID NO:3, and a LBD and, optionally, an N-terminal domain derived from another nuclear receptor, can be succesfully used to identify novel ligands or hormone analogs for said nuclear receptors. Such chimeric receptors are especially useful for the identification of the respective ligands of orphan receptors.
Since steroid receptors have three domains with different functions, which are more or less independent, it is possible that all three functional domains have been derived from different members of the steroid receptor superfamily.
Molecules which contain parts having a different origin are called chimeric. Such a chimeric receptor comprising the ligand-binding domain and/or the DNA-binding domain of the invention may be produced by chemical linkage, but most preferably the coupling is accomplished at the DNA level with standard molecular biological methods by fusing the nucleic acid sequences encoding the necessary steroid receptor domains. Hence, DNA encoding the chimeric receptor proteins according to the invention are also subject of the present invention.
Such chimeric proteins can be prepared by transfecting DNA encoding these chimeric receptor proteins to suitable host cells and culturing these cells under suitable conditions.
It is extremely practical if, next to the information for the expression of the steroid receptor, also the host cell is transformed or transfected with a vector which carries the information for a reporter molecule. Such a vector coding for a reporter molecule is characterized by having a promoter sequence containing one or more hormone responsive elements (HRE) functionally linked to an operative reporter gene. Such a HRE is the DNA target of the activated steroid receptor and, as a consequence, it enhances the transcription of the DNA coding for the reporter molecule. In in vivo settings of steroid receptors the reporter molecule comprises the cellular response to the stimulation of the ligand. However, it is possible in vitro to combine the ligand-binding domain of a receptor to the DNA binding domain and transcription activating domain of other steroid receptors, thereby enabling the use of other HRE and reporter molecule systems. One such a system is established by a HRE presented in the MMTV-LTR (mouse mammary tumor virus long terminal repeat sequence in connection with a reporter molecule like the firefly luciferase gene or the bacterial gene for CAT (chloramphenicol transferase). Other HRE""s which can be used are the rat oxytocin promotor, the retinoic acid responsive element, the thyroid hormone responsive element, the estrogen responsive element and also synthetic responsive elements have been described (for instance in Fuller, ibid. page 3096). As reporter molecules next to CAT and luciferase xcex2-galactosidase can be used.
Steroid hormone receptors and chimeric receptors according to the present invention can be used for the in vitro identification of novel ligands or hormonal analogs. For this purpose binding studies can be performed with cells transformed with DNA according to the invention or an expression vector comprising DNA according to the invention, said cells expressing the steroid receptors or chimeric receptors according to the invention.
The novel steroid hormone receptor and chimeric receptors according to the invention as well as the ligand-binding domain of the invention, can be used in an assay for the identification of functional ligands or hormone analogs for the nuclear receptors.
Thus, the present invention provides for a method for identifying functional ligands for the steroid receptors and chimeric receptors according to the invention, said method comprising the steps of
a) introducing into a suitable host cell 1) DNA or an expression vector according to the invention, and 2) a suitable reporter gene functionally linked to an operative hormone response element, said HRE being able to be activated by the DNA-binding domain of the receptor protein encoded by said DNA;
b) bringing the host cell from step a) into contact with potential ligands which will possibly bind to the ligand-binding domain of the receptor protein encoded by said DNA from step a);
c) monitoring the expression of the receptor protein encoded by said reporter gene of step a).
If expression of the reporter gene is induced with respect to basic expression (without ligand), the functional ligand can be considered as an agonist; if expression of the reporter gene remains unchanged or is reduced with respect to basic expression, the functional ligand can be a suitable (partial) antagonist.
For performing such kind of investigations host cells which have been transformed or transfected with both a vector encoding a functional steroid receptor and a vector having the information for a hormone responsive element and a connected reporter molecule are cultured in a suitable medium. After addition of a suitable ligand, which will activate the receptor the production of the reporter molecule will be enhanced, which production simply can be determined by assays having a sensitivity for the reporter molecule. See for instance WO-A-8803168. Assays with known steroid receptors have been described (for instance S. Tsai et al., Cell 57, 443, 1989; M. Meyer et al., Cell 57, 433, 1989).